Mechanisms of

Hemostasis
Maureane Hoffman
Professor of Pathology
she is a clinical
pathologist - she Duke University Medical Center
does blood banking/
laboratory
hematology, in case
Path & Lab Medicine Service
you were wondering
Durham VAMC email- maureane.hoffman@va.gov
maureane.hoffman@med.va.gov
286-0411 x6494
 Objectives
Understand how hemostasis relates
to the bodys response to tissue injury
Differentiate the newer cell-mediated
model from the classic cascade model
Describe the basic coagulation tests
and how they relate to the clotting
cascade
we will talk about the coagulation cascade and compare and
contrast this with newer cellular models of how coagulation works in
the body.
we will also highlight some defects/ things that can go wrong in
hemostasis.
 Coagulation:
when there is an injury,
Host Response to Injury
the body must mount a
response to halt
immediate damage, deal
with an infection, and
heal the wound and
restore tissue fxn. the
first step in this process
is coagulation which not
only stops bleeding, but
produces mediators such
as growth factors and
cytokines which help
condition and direct the
rest of this process...
 first step in hemostasis
involves platelets
Primary Hemostasis
Platelets Adhere & Activate at Sites of Injury

when there is an
injury or
inflammation, they
change shape and
bind to the
extracellular matrix
platelets are anuclete and to eachother.
fragments of cells that they can stop
circulate in the blood bleeding by
and are normally disc themselves, and
shaped. in this form they can express
they are not lipids on their
responsive and not surface upon
sticky. activation that
provide a good site
for the coagulation
reactions to take
place
 Secondary Hemostasis
point of secondary hemostasis is to consolidate
platelet plug in a fibrin meshwork

Coagulation proteins act on platelet surfaces to

form fibrin, which stabilizes the platelet plug

burried under here

are some activated
platelets

just fibrin

RBCs, leukocytes,
and platelets are
trapped in the
meshwork.
leukocytes will also
migrate in, and help
to disolve the clot
How can we
make sense of
hemostasis?
its complicated, but we will try to highlight
key features that will help us make sense
of things that happen in our pts
 In 1904 Paul Morawitz
proposed a model of
coagulation
"devil is in the
details of what that
thrombokinase is"

enzyme

the meshwork that

fibrinogen is converted we want to end up
to fibrin by thrombin with

Morawitz, P. Beitrge zur Kenntniss der Blutgerinnung Dtsch Arch

klin Med 1904;79:1-28
More and more factors were discovered and named different
things, and it all went down hill from there..
hemostasis was well studied
because of hemophelia in royal
families Hemophilia C factor
fibrinogen Labile Factor
prothrombin Laki-Lorand Factor
accelerator (AC-) globulin Pavlovsky Factor
Antihemophiliac Factor Plasma Thromboplastic Factor
Antihemophilic Factor B Plasma Thromboplastic Factor A
Antihemophilic Globulin (AHG) Plasma Thromboplastin Antecedent (PTA)
Antihemophilic Globulin A Plasma Thromboplastin Component
Autoprothrombin I Plasmakinin
Autoprothrombin II Platelet Cofactor
Autoprothrombin III Proaccelerin
Beta cothromboplastin Proconvertin
Christmas Factor Prothrombokinase
Contact Factor Protransglutamidase
Cothromboplastin Prower Factor
Facteur Antihemophilique A Robbins Factor
Fibrin Stabilizing Factor Serum Factor
Thromboplastic Plasma Component Serum Prothrombin Conversion Accelerator
Thromboplastinogen (SPCA)
Hageman Factor Stable Factor
Hemophilia A factor Stuart Factor
Hemophilia B Factor Stuart-Prower Factor
Thrombokatalysin
 In 1958 the International
Society on Thrombosis and
Hemostasis convened a
conference to standardize the
nomenclature

Thats how we got all those

roman numerals
at this point though, we still
didn't know how it worked -
this is why the roman
numerals are not in the right
order
 something in the tissue outside
the blood that promotes clotting
- we don't really call it factor III
anymore. we know now that it is

Coagulation Proteins
a specific protein called tissue
factor

Factor Synonyms Function Factor Synonyms Function

I Fibrinogen polymer unit IX Christmas factor, plasma protease
thromboplastin
II Prothrombin protease component
X Stuart factor, Stuart- protease
III Tissue cofactor
Prower factor
thromboplastin,
tissue factor XI Plasma thromboplastin protease
antecedent
IV Calcium we don't call Calcium factor
IV either... XII Hageman factor protease
V Accelerator globulin. cofactor
proaccelerin, labile released from platelets in a partially XIII Fibrin stabilizing Fibrin
factor active form, which was known as factor, fibrinoligase crosslinker
factor VI, but we don't call it that
VII Proconvertin, stable protease ---- Prekallikrein (Fletcher protease
factor factor)

VIII Antihemophilic factor cofactor ---- High-molecular- cofactor

or globulin weight kininogen
(Fitzgerald factor)
unless otherwise specified, we still
use the factor name and number to
identify parts of the cascade. :)

Factor VI was at one time used to designate activated Factor V.

 but nobody really knew
how all those factors interacted
to turn liquid plasma into a
solid fibrin clot
Thats why the roman numerals
arent in order in the coagulation
cascade - thus making it is hard
for us to remember
In the 1960s the coagulation
factors were organized into a
cascade or waterfall
model. This evolved into the
current cascade model
the idea of a sequence of proteases, acting as a
biological amplifier (apoptosis and complement also
work this way - but the coagulation cascade was
described first - this was a groundbreaking idea)
1. Macfarlane RG. An enzyme cascade in the blood clotting
mechanism, and its function as a biological amplifier. Nature.
1964;202:498-499.
2. Davie EW, Ratnoff OD. Waterfall sequence for intrinsic blood
clotting. Science. 1964;145:1310-1312
 Eventually the many
coagulation factors were
organized into a cascade
model
originally they thought it
looked like this - only one
pathway - but this isn't right Landmark
description
of
coagulation
as a
biological
amplifier

Earl W. Davie
Oscar D. Ratnoff

Science 1964;
145:1310-1312
 The cascade model evolved
into what my generation of
medical students was taught .
 aPTT = activated partial PT = prothrombin time
thromboplastin time

Intrinsic Pathway Extrinsic Pathway deficiency in one of the

aPTT PT factors will give you a
longer time in the aPTT
aPTT = long
abbreviation for test or the PT, depending on
the factor
Factor XII/HMK/PK = long pathway
contact factors, bind
to a charged
PT = short
surface, which
abbreviation for test
activates factor XII,
= short pathway
Factor XI Factor XIa which then activates
factor XI, etc

Factor IX Factor IXa Factor VIIa

Factor VIIIa Tissue Factor
on this slide -> factor III = tissue
top= protease, juice
bottom= cofactor
Factor X Factor Xa Factor X
Factor Va
these reactions almost surface for this rxn
always happen on a is a cell that has
phospholipid surface tissue factor on it Prothrombin Thrombin
surface - this is important outside the vessel
bc we don't want this rxn
to spread throughout the most of these
factors also require
body - we want them to
be localized to a surface Ca - this is impt bc if Fibrinogen Fibrin
we take out calcium,
hemopheliacs have a normal
we can decoagulate
PT - they have factor VIII or IX
blood
deficiencies
she basically read this slide:

Homologous Coagulation Factors

Vitamin K-Dependent Serine Proteases:
Factors II, VII, IX & X gene duplication
these guys are all very closely related- probably arose by

prothrombin
Structurally similar
Circulate as inactive zymogens
Activated by proteolysis
Work best in complex with a protein cofactor on
lipid surface containing phosphatidyl serine
Activity is calcium dependent
you do not need to be able to
recognize structures

some are activated by one

cleavage, others are activated
by two cleavages - these
cleavages allow them to
rearrange into an active
conformation

Roberts, Monroe & Hoffman: Molecular Biology and Biochemistry of the Coagulation Factors and
Pathways of Blood Coagulation. In Williams Hematology 7th ed, 2005
Why should I care about the
biochemistry of coag factors?
 Why should I care about the
biochemistry of coag factors?
It helps explain some things that are very useful
How does Coumadin (Warfarin) work?
How do calcium chelators act as one of the most

anticoagulants? widely prescribed

drugs out there,
even though its a
very old drug
 Things that are necessary for
coagulation proteases to work
Post-translational modification to produce
which gives them a
gamma-carboxy glutamic acid (Gla) negative charge

residues, which is vitamin K dependent

Calcium to bind to Glas and hold the
protein in the right conformation
active

Phospholipid surface for the proteases to

bind to along with their cofactors
Vitamin K-dependent factors
contain Gla-residues
3. warfarin inhibits
this reductase,
keeping the vitamin
K from being
reduced back to the
usable state.

2. vitamine k is
oxidized in this
reaction

4. pts given
warfarin take a
1. glutamates in the while to become
Gla domain get "anticoagulated"
caroboxylated by
carboxylase
 Warfarin: Commonly Used
Oral Anti-Coagulant
Warfarin alters synthesis of vitamin K-dependent factors
by preventing vitamin K-dependent carboxylation of
these proteins are
Factors II, VII, IX, X actually
anticoagulants and
Protein C & Protein S also get messed up
by warfarin

Result: no longer bind calcium they

so - they aren't in the right conformation and
can't bind phospholipids

New proteins must be synthesized to overcome the

warfarin effect

when people need their warfarin reduced

what do we do? - give them plasma
 Coag proteins work as
protease/cofactor complexes

little a = activated form

 Vitamin K-dependent proteases
(FII, VII, IX and X) need both tissue
factor and
calcium for
proper fxn

calcium forces these

hydrophobic guys to
the outside, and
holds the hydrophilic
Gla residues in the
inside, which allows
the factor to want to
bind phospholipid

phosphatidyl serine
Roberts, Monroe & Hoffman: Molecular Biology and Biochemistry of the Coagulation Factors
and Pathways of Blood Coagulation. In Williams Hematology 7th ed, 2005
 The Coagulation Cascade

Helps us interpret clinical laboratory tests

Prothrombin time (PT)
Activated Partial Thromboplastin Time (aPTT)
Intrinsic Pathway Extrinsic Pathway
aPTT PT
Factor XII/HMK/PK

Factor XI Factor XIa

Factor IX Factor IXa Factor VIIa

Factor VIIIa Tissue Factor

Factor X Factor Xa Factor X

Factor Va

Prothrombin Thrombin

Fibrinogen Fibrin
The Coagulation Cascade Doesnt Explain
How Blood Clots in vivo

Patients lacking FXII, HMK, or PK have a

long aPTT but no bleeding
Patients lacking FXI have a long aPTT and
may or may not have bleeding
Patients lacking FVIII or FIX have an
equally long aPTT and serious bleeding
 moral of the story:
aPTT and PT tests tell you if your pt is

Intrinsic Pathway deficient in one of these factors (but not

which one), but does not predict the bleeding
tendency of the patient - it happens
aPTT differently on cells in the body than it does in
a test tube.

Factor XII/HMK/PK Prolonged aPTT only

really long
no bleeding
tendency - so prob
no role in
Prolonged aPTT
hemostasis, more Factor XI Factor XIa Variable bleeding
likely to play a role
in inflamation,
probably in lysing
clots, and maybe in Factor IX Factor IXa Prolonged aPTT
thrombosis
Factor VIIIa Severe bleeding
not as long as if you
only severe are XII deficient
bleeding in some
sort of trauma or Factor X Factor Xa
surgery
Factor Va

Prothrombin Thrombin

Fibrinogen Fibrin
How does it really work
in the body?
Cells are important in the
body, but arent included
in the coagulation
cascade or the clinical
lab tests.
platelets are left out
in the lab bc they
are "a pain in the
rear to handle"

bc we donit have cells or platelets,

only some phospholipids and the
factors in lab tests - they are probably
not a very good model of reality
Hemostasis Occurs on Two Surfaces:
TF-bearing Cells and Platelets

1. Initiation
tissue factor bearing cell
initiates the coagulation
process by making a little
bit of thrombin, which then
activates platelets stuck
IIa down at the site of injury

2. Amplification 3. Propagation

IIa
platelets generate lots of
thrombin - the bulk of
thrombin responsible for
forming the fibrin clot
TF forms a hemostatic envelope
around the vessel

tissue factor is
stained brown here.
it surrounds a vessel
so that when the
vessel is disrupted,
blood can come in
contact with tissue
factor and activate
the extrinsic pathway
 Initiation
X
Xa
TF
VIIa TF VIIa

Tissue factor bearing cell can

activate factor X and also factor
TF-Bearing Cell IX, which generates a little
thrombin but that doesn't do
much normally

TF

VIIa
IX

IXa

Hoffman M, Monroe DM: A Cell-Based Model of Hemostasis. Thromb Haemostas, 85:958-65, 2001
however, when factors are
there, the thrombin can help
activate the platelets. this
Amplification
X
priming amt of thrombin helps
set the stage for the platelets
to help take over the
coagulation process

VIIa
II
Xa VIII/vWF VIIIa+Free vWF
TF TF VIIa
Va
IIa XI

TF-Bearing Cell V Platelet

V XIa
Va
TF
Priming Amount
VIIa of Thrombin

VIIIa
XIa
Va

Activated Platelet
 Propagation
VIII/vWF VIIIa + Free vWF
TFPI Xa
Xa VIIa VIIa XI
TF TF
Va IIa

V Platelet
V XIa
TF-Bearing Cell
Va
then, the platelets, using primarily the
intrinsic pathway, generates a whole bunch
TF of thrombin, and we no longer need the TF-
bearing cell

VIIa
IX X
II Large amount
of thrombin
IXa
IX Xa
IIa
XIa VIIIa
Va

Activated Platelet
 Fibrin Clot Formation

then that thrombin converts

fibrinogen to fibrin
IIa

Fibrinogen
fibrin forms
polymers that are
cross linked by FIBRINOGEN
factor XIII

D E D
THROMBIN

FIBRIN MONOMER

POLYMERIZATION

POLYMERIZING FIBRIN
 Factor XIII

Activated by thrombin during coagulation

Has transglutaminase activity
Covalently crosslinks fibrin strands to
stabilize the clot
 A Cell-Based Model of
Hemostasis
same thing we just saw, except now in a
pink cartoon version:

X II
VIII/vWF
TF VIIa Xa Va IIa
TF-Bearing Cell VIIIa

TF VIIa V Va XI XIa
IX
Platelet
IXa X II
problem area in
Xa IIa
IXa VIIIa
hemophelia. you get
initiation, but the Va
platelet thrombin XIa
generation "fizzles" Activated Platelet

IX
Hoffman M, et al. Blood Coagul Fibrinolysis. 1998;9(suppl 1):S61-S65.
 PT: measures extrinsic/initiation
pathway*

*short name (PT) = short pathway

Monroe, DM and Hoffman, M: What does it take to make the perfect clot? Arterio Thromb Vasc Biol 26:41-48, 2006
 aPTT: measures intrinsic/platelet
pathway* aPTT measures the pathway
that happens on the platelet
surface

*long name (aPTT) = long pathway

Monroe DM and Hoffman, M: What does it take to make the perfect clot? Arterio Thromb Vasc Biol 26:41-48, 2006
 The intrinsic and
extrinsic pathways are
not redundant,
but have distinct roles in
hemostasis in vivo
so if your patient is missing components of
either pathway, they can have a bleeding
problem
 when the wound heals and we
have to dissolve the fibrin clot =
fibrinolysis Fibrinolysis
TISSUE PLASMINOGEN ACTIVATOR (tPA)
activates used to dissolve
plasminogen to STREPTOKINASE clots in people,
plasmin and is UROKINASE comes from bacteria
released by
endothelial cells. FACTOR XIIa found in urine,
certain vascular released by WBCs
beds produce a lot KALLIKREIN
of tPA if they tend to
get a lot of clots - ex:
lungs PLASMINOGEN
contact factors that used to block
don't play act in binding of tPA to
hemostasis but play plasminogen and
a role in converting plasminogen to fibrin
plasminogen to in pts who are
plasmin bleeding.

active enzyme
PLASMIN no longer on the
market, but inhibited
FIBRIN
plasmin
FIBRIN CLOT
FRAGMENTS
 FIBRINOGEN

D E D
+ 2 FRAGMENTS PLASMIN
A,B,C

FRAGMENT X

D E D plasmin cleaves the

rod of fibrinogen
and degrates the
fibrin clot into
PLASMIN smaller pieces.

FRAGMENT Y FRAGMENT D

D E D
PLASMIN

FRAGMENT D FRAGMENT E

D E
 Control of Clot Formation
in the modern world, we
are a lot more likely to clot

Separation of Initiation & Propagation to death than to bleed to

death... so its important to
extrinsic pathway and platelet are separated by the vessel wall - only get large scale know how to deal with clots
thrombin generation if these interact that are forming in the

Presence of plasma coagulation inhibitors

wrong places.

Antithrombin (AT or ATIII) inhibits almost all of the coagulation

proteases to some degree

Activity increased by heparin & LMWH people deficient in AT have

thrombosis, people deficient
Tissue Factor Pathway Inhibitor (TFPI) tissue factor inhibitor aren't
born - knockout mice dont
survive if they are born

Anti-thrombotic mechanisms on healthy vascular

endothelial cells
Thrombomodulin (TM)/Protein C/S system
vitamin K dependent
- degrade activated
coagulation factors
Heparan sulfates that bind AT so thrombin is not
made on healthy
endothelial cells
 Hemostasis Sets the Stage for
Inflammatory Cell Influx & Effective Wound Healing

Thrombin Fibrin is Platelets Hemostatic

has many the matrix release defects can
biological for cytokines lead to
activities healing & growth defective
set the stage for make scaffold for
factors wound
healing healing to occur on
healing
 Bleeding Disorders
We are only going to talk about
inherited (not acquired) disorders
Platelet problems
Coagulation factor problems
 Clinical Bleeding
Platelet Problem
Petechiae & purpura small areas of bleeding

Mucocutaneous bleeding
Coagulation Factor Problem
Bruises (ecchymoses) big areas of bleeding

Soft tissue hemorrhage

"for your reference: platelets have all sorts of stuff in
them"
 Descriptors of Bleeding

Petichiae are < 3 mm,

Purpura are 0.3-1 cm,
Ecchymoses are > 1 cm

pinpoint
hemorrhages
characteristic of
"platelet bleeding"
they dont blanch
when you press on
them

http://www.uptodate.com
endothelial cells
Thrombocytopenia leads to
need platelets to
keep them happy
and healthy
endothelial changes

The top shows an EM of a

capillary from a thyroid
perfused with PRP for 5h
PRP = platelet rich
plasma

The bottom shows a capillary

from a thyroid perfused with
PPP for 5h. Note the disruption
in the endothelium. PPP = platelet poor
plasma
endothelial cells
retract or fall off Gimbrone et al: Preservation of vascular
integrity in organs perfused in vitro with a
platelet-rich medium. Nature, 222:13-4,
1969
 Thrombocytopenia leads to
endothelial changes
with PPP - this can happen:

This picture shows a RBC

extravasating from a capillary
of a thrombocytopenic
mouse (arrow)
RBC appear to traverse
small channels in the
endothelial cells.
Aursnes & Pedersen: Petechial
hemorrhage in the ciliary process of
thrombocytopenic rabbits. An EM
study. Microvascular Res, 17:12-21,
this does NOT happen in hemophilia or 1979
some other coagulation factor
abnormality
 Factor Deficiencies:
General Considerations
Deficiencies of each of the following exist:
deficiency of tissue
Factor VIII
hemophilia A
Factor IX
hemophilia b
factor does not exist -
you can't survive
without tissue factor.

Factor XI Factor VII vascular system will

not develop normally
in the absence of TF
or its inhibitor
Prothrombin Factor X
Factor V Factor XII
Fibrinogen Factor XIII
 Factor Deficiencies:
General Considerations
Inheritance: Most are inherited as
autosomal recessive disorders
Factors VIII and IX are encoded on the
X chromosome and their deficiencies
are sex-linked recessive
While bleeding is the hallmark of these
disorders, its severity and pattern vary
depending on the involved factor
 Congenital Bleeding Disorders
vonWillebrand Disease
Hemophilia A & B
FXI deficiency
vonWillebrand Disease
 vonWillebrand Disease
we will see this. it has a wide range of clinical
manifestations. may not show up until surgery,
trauma, or if the pt begins taking an anticoagulant.

Autosomal can also be very severe bleeding

Most common inherited bleeding disorder

vWF mediates platelet adhesion under high
shear - bleeding is typical of platelet defects petichiae

vWF is the carrier for FVIII - FVIII level may

be reduced and aPTT may be prolonged
Subdivided into several types based on
multimer pattern and antigen level
Hemophilia A & B

Deficiency of FVIII or FIX

 Hemophilia A and B
X-linked
Up to 30% from de novo mutation i.e. no
family history
Mild, moderate and severe forms
Dysfunctional molecules Cross-reacting
especially true with factor IX
material positive (CRM+)
Reduced level of a normal molecule -
Cross-reacting material negative (CRM-)
Intrinsic Pathway Extrinsic Pathway
aPTT PT
Factor XII/HMK/PK

Factor XI Factor XIa

Factor IX Factor IXa Factor VIIa

Factor VIIIa Tissue Factor

Factor X Factor Xa Factor X

Factor Va

Prothrombin Thrombin

Fibrinogen Fibrin
Hemophilia Is a Failure of Platelet
Surface Thrombin Generation
X II
may form a platelet plug
TF VIIa Xa Va IIa initially, but a few hours
later the would will
rebleed and wont stop
TF-Bearing Cell

TF VIIa V Va
IX
Platelet
IXa X II
IXa Va
Activated Platelet

Hoffman M, et al. Blood Coag Fibrinolys. 1998;9(suppl 1):S61-S65.

 Much early coagulation
research was driven by the
presence of hemophilia in the
royal families of Europe
she probably
suffered a denovo
mutation
 Inheritance of Hemophilia
Hemophilia A (FVIII deficiency) 1 in
10,000 live male births
Hemophilia B (FIX deficiency) 1 in
30,000 live male births
Inherited as sex linked recessive traits
An affected male will produce only normal males and
carrier females (with a normal female)
A carrier female will produce offspring of which half the females
are carriers and half the males are affected (with a normal male)
Inheritance of Hemophilias

all daughters will

carry

half of daughters will 50% of sons

carry affected
 Hemophilia A and B

Clinical picture is identical in A & B

Prolonged aPTT in both, need factor
assays to distinguish
Severely affected have spontaneous soft
tissue and joint hemorrhage
Severely deficient may develop antibody
inhibitors
FXI deficiency
long aPTT but bleeding isn't as bad as hemophilia
Intrinsic Pathway
aPTT

Factor XII/HMK/PK Prolonged aPTT only

Prolonged aPTT
Factor XI Factor XIa Variable bleeding

Factor IX Factor IXa Prolonged aPTT

Factor VIIIa Severe bleeding

Factor X Factor Xa
Factor Va

Prothrombin Thrombin

Fibrinogen Fibrin
 FXI Deficiency populations where
you have
consanguinity bc
that has a tendency
to concentrate
recessive genes

Autosomal
Common in certain populations -
Ashkenazi Jews, some Arab
populations a specific factor X deficieny has been traced to consanguinity
in the mountains of North Carolina.

Bleeding with trauma or surgery,

otherwise its usually mild
especially if on aspirin
Bleeding risk not predictable from
aPTT or FXI level